Pathways to improved polyene antimicrobial agents (PIPA)

天然产品是由植物和微生物制造的分子，它们启发了我们今天所依赖的许多重要药物的发展。例如，土壤细菌，如放线杆菌家族的链霉菌，在产生抗菌剂方面特别多产，可以杀死细菌、真菌和其他微生物病原体。其中许多天然抗菌剂已被广泛用于治疗危及生命的传染病。然而，由于抗菌素耐药性(AMR)，现有的抗菌药正变得越来越无效，微生物病原体迅速进化出逃避这些化合物作用的方法。真菌感染可能特别成问题，曲霉病和隐球菌病等疾病每年导致数百万人受到威胁生命的感染。直到最近，很明显，许多新冠肺炎患者(特别是在印度)死于由“黑真菌”毛霉病引起的继发感染。不幸的是，目前只有少量的抗真菌药物可供选择，而有希望开发的候选药物也寥寥无几。此外，目前使用的许多抗真菌药物在很大程度上对新出现的多重耐药真菌病原体无效，如对全球健康构成重大威胁的金黄色念珠菌。目前最有效和使用最广泛的抗真菌药物是多烯类两性霉素B、制霉菌素和苯丙氨酸，它们都是世卫组织的基本药物。多烯类化合物是从放线菌中提取的复杂天然产物，具有相似的大环结构。它们表现出极好的广谱抗真菌活性，而且与其他抗真菌药物不同，对多烯类药物的耐药性不那么普遍。尽管具有良好的性质，但聚烯类化合物的溶解度低，并显示出毒性。这是因为多烯结合并破坏真菌病原体的细胞壁(膜)，但也可以结合到人类细胞膜上，导致毒性效应。多烯类化合物以前是通过化学合成的方法制备的，毒性降低，溶解性增加。然而，这通常需要费力和昂贵的多步骤合成过程，这些合成过程不可持续，污染严重，放大生产成本太高。最近，我们已经对几种放线菌的基因组进行了测序，并发现了编码酶(催化剂)的基因，这些酶(催化剂)是合成(组装)新型多烯所必需的。我们能够分离和确定新的多烯的结构。此外，我们还获得了一些在多烯生物合成后期增加关键功能的“剪裁”酶的初步特征。在这个项目中，我们的目标是进一步表征新的裁剪酶，用天然底物(生物合成中间体)以及从其他途径到已知多烯的前体(两性霉素B等)来测试它们。我们还将获得结构或模型，我们可以用来突变(工程)裁剪酶，以扩大其底物范围，并促进催化替代裁剪反应。然后，我们的目标是将不同的剪裁酶在反应中结合起来，创建一个多样化的多烯衍生物文库，并对其进行抗真菌活性、毒性和溶解性测试。这将提供详细的结构-活性关系(SAR)，使我们能够确定哪种剪裁反应组合可以提供最佳性能的聚烯。最初，我们将使用分离的酶(在体外)生产新的多烯用于测试。然而，我们也将开发体内工程方法来创造能够在一步发酵中产生最好的多烯衍生物的放线菌菌株。这些基于生物的方法，特别是体内发酵，可以提供更可持续、更有效和更具成本效益的途径来改进多烯抗真菌药物，我们迫切需要这些药物来对抗新出现的耐药真菌病原体。

Natural products are molecules made by plants and microorganisms that have inspired the development of many important pharmaceuticals that we rely on today. For example, soil bacteria, such as Streptomyces from the Actinobacteria family, are particularly prolific in producing antimicrobial agents, which can kill bacterial, fungal, and other microbial pathogens. Many of these natural antimicrobial agents have been widely used to treat life-threatening infectious diseases. However, existing antimicrobial drugs are becoming increasingly ineffective due to antimicrobial resistance (AMR), with microbial pathogens rapidly evolving ways to evade the effects of these compounds. Fungal infections can be particularly problematic, with conditions such aspergillosis and cryptococcosis resulting in millions of life-threatening infections every year. Only very recently, it became apparent that many COVID-19 patients (particularly in India) died of a secondary infection, caused by the "black fungus" mucormycosis. Unfortunately, there are only a small number of antifungal drugs available and very few promising drug candidates in development. Moreover, many antifungals currently in use are largely ineffective against emerging multidrug-resistant fungal pathogens such as Candida auris which pose a major threat to global health. Currently the most effective and widely used antifungals are the polyenes amphotericin B, nystatin and pimaricin, which are all WHO essential medicines. Polyenes are complex natural products derived from Actinobacteria, with similar macrocyclic structures. They show excellent broad spectrum antifungal activity and, unlike other antifungal drugs, resistance to polyenes is less widespread. Despite possessing favourable properties, polyenes suffer from low solubility and exhibit toxicity. This is because polyenes bind to and disrupt the cell wall (membrane) of fungal pathogens, but can also bind to the human cell membrane, leading to toxic effects. Polyene derivatives with reduced toxicity and increased solubility have previously been prepared by chemical synthesis. However, this typically requires laborious and expensive multistep synthetic procedures, which are unsustainable, polluting and too costly to scale-up for manufacture.Recently, we have sequenced the genomes of several Actinobacteria and discovered genes encoding enzymes (catalysts) that are required for the biosynthesis (assembly) of novel polyenes. We were able to isolate and determine the structure of new polyenes. In addition, we obtained preliminary characterisation for some "tailoring" enzymes that add key functionality during the latter stages of polyene biosynthesis. In this project, we aim to further characterise the new tailoring enzymes, testing them with natural substrates (biosynthetic intermediates) as well as precursors from other pathways to known polyenes (amphotericin B etc.). We will also obtain structures or models which we can use to mutate (engineer) the tailoring enzymes to broaden their substrate scope and facilitate catalysis of alternative tailoring reactions. We then aim to combine the different tailoring enzymes in reactions to create a diverse library of polyene derivatives, which will be tested for antifungal activity, toxicity, and solubility. This will provide a detailed structure-activity relationship (SAR), enabling us to establish which combination of tailoring reactions provides polyenes with the best properties. Initially, we will use isolated enzymes (in vitro) to produce new polyenes for testing. However, we will also develop in vivo engineering approaches to create Actinobacterial strains that can produce the best polyene derivatives in a single-step fermentation. These bio-based approaches, particularly in vivo fermentation, can provide much more sustainable, efficient, and cost-effective routes to the improved polyene antifungal agents that we urgently need to combat the emerging drug-resistant fungal pathogens.